Methods of delivery of exogenous proteins to the cytosol and uses thereof

ABSTRACT

The present invention is directed to a method for delivering exogenous proteins to the cytosol, by binding a target antigen (such as a protein) to a transport factor that contains a fragment of a bipartite protein exotoxin, but not the corresponding protective antigen. Preferably, the target antigen is fused to the transport factor. Preferred transport factors include the protective antigen binding domain of lethal factor (LFn) from  B. anthracis , consisting of amino acids 1-255, preferably a fragment of at least 80 amino acids that shows at least 80% homology to LFn, and a fragment of about 105 amino acids from the carboxy portion that does not bind PA. The target antigen can include any molecule for which it would be desirable to elicit a CMI response, including viral antigens and tumor antigens.

This application is the 35 U.S.C. §371 entry of PCT/US02/09680, whichclaimed benefit under 35 U.S.C. §119(e) of 60/279,366 filed on Mar. 28,2001.

This invention was supported by National Institutes of Health grantAI47539 and the government of the United States has certain rightsthereto.

FIELD OF THE INVENTION

The present application is directed to a method of delivering exogenousproteins to the cytosol, novel fusion proteins and uses thereof.

BACKGROUND OF THE INVENTION

Much attention has focused on methods for generating immune reactions.One class of immune reaction to foreign antigens is the production ofantibodies, typically referred to as humoral immunity. A second form ofimmune reaction results from the presentation of antigen by an antigenpresenting cell (APC). This type of immune reaction is broadly referredto as cell mediated immunity (CMI), or T cell responses. Although bothtypes of immune responses are important, considerable attention hasrecently focused on CMI. In dealing with infectious diseases such asAIDS, caused by the Human Immunodeficiency Virus (HIV), the antibodyresponses to the virus and portions thereof have not proven sufficientto confer immunity. Similarly, in dealing with exogenous proteinsassociated with many malignancies, the antibody responses have also notproven sufficient. Thus, speculation has focused on generating CMIresponses.

In order to elicit CMI, an antigen must be bound to a majorhistocompatibility complex (MHC) class I or II molecule on the surfaceof the APC. The class I molecules typically present antigens externally,such as endogenous proteins, those from viral infections, and tumorantigens. Antigen-specific T cells typically recognize infected targetcells when the pathogen-derived (or cancerous) peptide epitopes (usually8 to 10 amino acids) are presented by molecules encoded by the hostclass I MHC (7). These epitopes are derived from cytoplasmic proteinscleaved by the proteosome into small peptide fragments. These are thentransported into the lumen of the endoplasmic reticulum (ER), where theycomplex with newly synthesized MHC-I molecules and are subsequentlytransported to the cell surface, where recognition by T cells occurs(8-13). Antigens in the extracellular fluid (exogenous antigens)generally do not gain access into this processing compartment in mostcells. Thus, a significant challenge to eliciting CMI with a vaccine isthe delivery of exogenous antigens to the cytosol for presentation byMHC class I molecules. It would be desirable to be able to generatevaccines to a wide variety of infectious diseases, such as HIV, as wellas cancers, such as prostate cancer, breast cancer and melanoma.

For example, growing evidence suggests that CMI plays an essential rolein controlling HIV infection (Ogg et al., Science 279:2103-6 (1998);Schmitz et al., Science 283:857-60 (1999); Brodie et al., Nat. Med.5:34-41 (1999)). Individuals who have been exposed to HIV but remainuninfected often have antiviral CMI but no antibody response. Theviremia of primary infection resolves as viral specific cytotoxic Tlymphocytes (CTL) develop, before the development of specific antibodies(Letvin, Science 280:1875-96 (1998)). These data illustrate the centralrole CMI plays in controlling HIV infection.

Many tumors are associated with the expression of a particular proteinand/or the over-expression of certain proteins. For example, prostatecancer is associated with elevated levels of protein such as ProstateSpecific Antigen (PSA). Breast cancers can be associated with theexpression and/or over-expression of protein such as Her-2, Muc-1, CEA,etc. Thus, considerable attention has been aimed at trying to generateimmune responses, particularly developing CMI, to such antigens in thetreatment of such malignancies.

Approaches to developing cell mediated immunity to infectious diseaseshave included using the entire infectious agent, for example, by makinggenetically engineered inactivated viruses or using a killed infectiousagent. Another approach has been subunit vaccines, which is presentingone or more antigens (but not the entire virus) to a subject.

In order to generate CMI, antigen must be delivered to the interior ofthe cell. Exogenous proteins are poorly taken up by the cell.Accordingly, the preferred method has been using procedures such asviral vectors, liposomes, naked DNA or a similar approach. However, suchapproaches have many draw backs. For example, many recombinant virusesgenerate antigenic reactions themselves, upon repeated administration.Since standard forms of generating immune reactions typically require aninitial injection, referred to as the prime, and subsequent injections,referred to as boosts, to achieve a satisfactory immunity, this can be aserious problem. Moreover, while much attention has been placed onimproving the safety of viral vectors, there are always certain risks.For example, many of the target populations, such as those infected withHIV, may have a weakened immune system. Thus, certain viral vectors thatare perfectly safe in many individuals may pose some degree of risk tothese individuals. Methods of delivering protein to cells have also notproven entirely satisfactory as of this time. Accordingly, there is aneed for new and simple methods to deliver an antigen to the cytosol tostimulate CMI.

In trying to develop CMI responses, there have also been technicalproblems with the difficulty in measuring these responses.

Current available laboratory assays to detect cell-mediated immuneresponses have serious shortcomings, especially when applied to largevaccine efficacy trials in various clinical settings. This is becausethe available equipment and technical support required to measure CMIusing current techniques are often minimal in the setting where they arerequired, in the field. CTL are thought to play a crucial role incontrolling HIV-1 infection, and many HIV-1 vaccine candidates aredesigned to stimulate T cell responses as well as neutralizingantibodies (1-6). However, the standard laboratory methods for detectingCMI, such as HIV-specific CTL, are complex, time consuming, and oftenrestricted to highly specialized facilities. An improved method formeasuring T cell responses will have a significant effect on thedevelopment of all T cell dependent vaccines or immune therapies. Thisstrategy can potentially be applicable to other fields of research whereCMI responses are known to play an important role in prevention andcontrol of the diseases.

One difficulty in reliably detecting CMI response in vitro results fromthe unique requirement for antigen presentation. As described above, thedelivery of exogenous proteins to the cytosol for presentation to Tcells by MHC class I molecules represents a significant challenge. Thisphysical partition of the class I pathway has been a major barrier todetect T cell responses in vitro. Consequently, most of the currentlaboratory methods in measuring CMI utilize live viral or bacterialvectors to deliver antigens into cytosol, among which recombinant poxsuch as vaccinia viruses are the most commonly used. Another approach isto externally load MHC-I molecules on surface of target cells withsynthetic peptides (10 to 20 amino acids) derived from known CTLepitopes. These methods have serious limitations in general clinicaluses. The use of a live viral vector, such as a recombinant vacciniavirus, requires trained and immunized laboratory personnel, includingminimum containment facilities as precautionary safety measures.Synthetic peptides are not only prohibitively expensive, but the designof the “universal” peptide profile that fit the diversified MHC-Imolecules in various populations is extremely difficult. The challengefor the development of assays to measure T cell responses is, therefore,to deliver large pieces of exogenous antigens into cytosol withoutresorting to live recombinant viral or bacterial vectors.

Accordingly, it would be desirable to have kits that could be used formeasuring CMI in vitro. It would be particularly desirable to have kitsthat could be readily used in remote locations such as Africa, India andAsia, where there are many proposals to test a number of vaccinecandidates such as vaccines against HIV.

We have now discovered that a family of bipartite protein exotoxins,such as Bacillus anthracis, contains fragments that can be used for thedelivery of exogenous antigens, such as proteins, to the cytosol. Onepreferred protein fragment from these proteins is from the N-terminalportion that contains the protective antigen (PA) binding domain, butnot those portions resulting in toxicity to the cell. More preferably,that fragment has been modified to remove the specific domain that bindsto PA.

B. anthracis is the causative agent of anthrax in animals and humans.The toxin produced by B. anthracis consists of two bipartite proteinexotoxins, lethal toxin (LT) and edema toxin. LT is composed ofprotective antigen (PA) and lethal factor (LF), whereas edema toxinconsists of PA and edema factor (EF). None of these three components,PA, LF, and EF, alone is toxic. Once combined however, edema toxincauses edema and LT causes death by systemic shock in animals andhumans. Consistent with its critical role in forming both toxins, PA hasbeen identified as the protective component in vaccines against anthrax.The molecular mechanism of anthrax toxin action is currentlyhypothesized as follows: PA is a 735-amino acid polypeptide that bindsto the surface of mammalian cells by cellular receptors. Once bound, PAis activated by proteolytic cleavage by cellular proteases to a 63-kDamolecule capable of forming a ring-shaped heptamer in the plasmamembrane of the targeted cell (FIG. 1) (6, 7). The PA heptamer thenbinds either EF or LF, which are internalized by endocytosis. Afterendosomal acidification, PA enables EF or LF to enter the cytosol,presumably by means of a pore formed by the heptamer. Within thecytosol, EF acts as an adenylate cyclase (8) to convert ATP to cAMP.Abnormally elevated levels of cAMP perturb cellular metabolism.

The action of LF in the cytosol causes the death of host cells by amechanism that is not well understood. LF induces over-production of anumber of lymphokines (9), contributing to lethal systemic shock in hostanimals. Recent studies also show that LF has two enzymatic activities:it can act as a zinc metalloprotease (10), and it inactivates themitogen-activated protein kinase (11). Although it is still not clearhow these two enzymatic activities of LF are connected, both arerequired for LF toxicity. It has previously been reported that anthraxtoxin B moieties may be used to deliver eptiopes which in turn elicit anantibody response by the immune system, in the presence of PA (WO97/23236).

LF is a 776 aa polypeptide, and the functional domain for both enzymaticactivities is located between amino acids 383 and 776 of LF (FIG. 2A;SEQ ID NO: 1). The N-terminal truncated LF (LFn) polypeptide is a 255amino acid polypeptide (corresponding to residues 34-288 of SEQ ID NO:2). The 255 amino acid LFn polypeptide part is derived from a precursorprotein of 1-288 residues as shown in FIG. 2B (SEQ ID NO:2), where thefirst 1-33 amino acids correspond to the signal peptide. Without thecatalytic domain, LFn polypeptide (residues 34-288 of SEQ ID NO: 2)completely lacks any toxic effect when mixed with PA and added tocultured macrophages or when injected into animals. It does, however,still bind to PA effectively. The PA binding domain of the LFnpolypeptide is located within the first 1-149 N-terminal amino acids ofthe LFn polypeptide (i.e. where the LFn polypeptide is residues 34-288of SEQ ID NO: 2, thus the first 1-149 N-terminal amino acids of the LFnpolypeptide are residues 34-184 of SEQ ID NO:2.

SUMMARY OF THE INVENTION

The present invention provides methods of delivering exogenous antigensto the cytosol, novel fusion proteins, and uses thereof.

We have now further found that one can use a transport factor that islethal factor modified to inactivate toxin domains, fragments thereofsuch as LFn and fragments of LFn, such as a fragment containing thecarboxy portion of that fragment, as a transport factor, fused to atarget antigen, without PA to deliver the antigen to the cytosol.Preferably, the transport factor is LFn or a fragment thereof. Onepreferred group is LFn fragments do not contain the PA binding domain.More preferably, the transport factor is an LFn fragment. For example,the 60 carboxy most amino acids of LFn can be used as a transportfactor, still more preferably the 80 carboxy-most amino acids. One canalso use other fragments. For example, one can use fragments containingmore of the lethal factor protein as long as one inactivates the toxinportion. Preferably the transport factor fragment contains a portion ofthe 80 carboxy-most amino acid residues of LFn and contains otherportions of the fragment as long as those portions containing toxicityare removed. Preferably the fragment is 350 amino acids or less, stillmore preferably it is 300 amino acids or less, even more preferably itis 250 amino acids or less. One preferred fragment is 105 amino acids orless. A more preferred fragment is 80 amino acids or less. Thistransport factor is then linked to the antigen you wish to bring to thecytosol. This can be done by techniques well known in the art. Forexample, one could prepare fusion proteins containing the antigen orantigens that one wants to bring to the cytosol of the cell.

The preferred methods of the invention are characterized by novelpolypeptides which elicit in treated animals the formation of a cellmediated immune response.

This invention provides DNA sequences that code for the novel fusionpolypeptides of the invention, recombinant DNA molecules that arecharacterized by those DNA sequences, unicellular hosts transformed withthose DNA sequences and molecules, and methods of using those sequences,molecules and hosts to produce the novel polypeptides and CMI immuneresponse to desired antigens this invention.

In another preferred embodiment, this invention provides apharmaceutical composition comprising one or more novel fusion peptidesof this invention. Such a composition is effective in eliciting cellmediated immune responses. In one preferred embodiment it can be used asa vaccine. In another embodiment, these fusion proteins can be used tocreate producer cells, preferably bacterial producer cells for a varietyof proteins, particularly proteins that have proven difficult to expressin such cells.

In another preferred embodiment, this invention provides a method formeasuring cell mediated immune responses.

In a further preferred embodiment, this invention provides a kit formeasuring cell mediated immune responses in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing depicting the PA-mediated entry of LFn into a cellvia endocytosis, and subsequent presentation by MHC Class I molecules.

FIGS. 2A-C show the amino acid sequence of various lethal fragment (LF)polypeptides. FIG. 2A shows the full length amino acid sequence of LF,with the first 1-33 residues constituting the signal peptide, and the LFpolypeptide beginning at residue 34 (SEQ ID NO:1). FIG. 2B shows theprecursor LFn protein of 1-288 residues, with residues 1-33 constitutingthe signal peptide, and residues 34-288 constitute the LFn polypeptide.(LFn corresponds to residues 34-288 of SEQ ID NO:2 and the precursor LFnprotein corresponds to residues 1-288 of SEQ ID NO:2). FIG. 2C shows thesequence of amino acids 185-288 of lethal factor, which corresponds toSEQ ID NO:3 herein and is sometimes referred to as Fragment 3.

FIGS. 3 A-B are graphs that show that LFn presentation of targets isPA-independent. Well characterized CTL clones were tested for activityin a standard chromium release assay at effector to target (E:T) 10:1.Targets were HLA-matched EBV-transformed cell lines that were sensitizedovernight with LFN in the presence or absence of PA. Controls used wererecombinant vaccinia vectors (NYCBH for control and rVV-Nef or Envpeptide for appropriate clone). FIG. 3A shows activity of a Nef-specificclone (KM) was tested against B60 restricted target cells incubatedovernight with Nef recombinant vaccinia vectors, PA and LFnNef or LFnNef alone. Background/control for each target was included (control,PA-LFn, LFn). FIG. 3B shows Env-specific CTL clone (SP 511) recognizestargets labeled with LFnEnv in a dose dependent fashion and was againPA-independent Control used were peptide Env 106B (100 μg/ml). Dose ofLFn construct is listed as μg/ml (LfnEnv 5=5μ/ml of LfnEnv).

FIGS. 4 A-B are graphs that show that LFn-HIV recognition isHLA-restricted. 4A shows HLA-B14 restricted Gag clone (AC13) was testedfor activity using HLA matched and mismatched B-LCL sensitized withLFn-p24 and tested in standard chromium release assay. Rvv-Gag was usedas control. 2B shows HLA-restriction for B60 Nef clone (KM) wasdemonstrated using HLA matched and mismatched targets sensitized withLFnNef. TAP-deficient (T2), LA-B60 target cells were sensitized withLFN-Nef and the optimal Nef peptide and tested for lysis. Lack ofactivity with T2 LFnNef target cells suggests requirement for proteasomeprocessing. All clones were used at E:T 10:1 and lysis level subtractedfrom those of background controls. NYCBH was used for recombinantvaccinia virus control and LFn alone for LFnP24 and LFnNef. Positivecontrol included Nef peptide 180 and recombinant vaccinia vector Gag(rvv-Gag).

FIGS. 5 A-B are graphs that show the internalization and processing ofLFn-HIV. 5A shows Nef-specific clone (KM) lysis of HLA-matched targetcells sensitized with LFnNef in the presence of brefeldin A,cytochalasin B and chloroquine compared to LFnNef alone. Co-incubationwith cytochalasin B and brefeldin A abrogated the recognition by aNef-specific clone but not with chloroquine. 5B shows activity of thesame Nef clone with target infected overnight with recombinant vacciniavirus expressing Nef or sensitized with the optimal epitope peptide Nef180 alone or in the presence of chloroquine, cytochalasin B or brefeldinA. No significant decrease in activity was seen when target cells andCTL clone were incubated in the presence of cytochalasin B, chloroquineor brefeldin A. Lysis levels shown are subtracted from background lysiswhich were less than 10%.

FIG. 6 are graphs that show LFn-HIV expression in the Elispot assay.Cryopreserved PBMCs from HIV-1 infected individuals with knownHIV-specific CTL activity or epitopes were used in the Elispot assayusing recombinant vaccinia viruses, or LFn-HIV. LFn alone and NYCBH wereused as control. Duplicate wells were used at 100K cell/well. Resultsare reported as SFC/million (spot forming cells per million).

FIGS. 7 A-B are FACS plots that show LFn-HIV expression in intracellularflow-based assay. Intracellular production of IFN-γ in Nef-specificclone (KM)(A) as well as from PBMC of a seropositive individual (AC2)with Env-specific CTL activity (B). Unstimulated cells were used asnegative control (1) and recombinant vaccinia viruses as positivecontrol (2) LfnNef and LfnEnv were used respectively (3).

FIGS. 8A and B are drawings depicting LFn fusion proteins.

FIG. 9 is a series of drawings depicting LFn fusion proteins.

FIG. 10 shows cellular uptake of LFn-GFP into CHO cells. CHO cells werecultured in collagen treated chamber slides to reach 80% confluence andwere then incubated with purified LFn-GFP for one hour, followed bywashing extensively with PBS and PBS plus proteases to remove membranebound proteins. The cells were then stained with the anti-transferrinantibodies and fixed with paraformaldehyde. The slides were examined byconfocal microscopy for green color (GFP, shown in FIG. 10 A-D) and redcolor (anti-transferrin, shown in FIG. 10 E-H), respectively. A thirdimage is presented for each field by super-expose the green image withthe red image of the same cells (FIG. 10 I-L). Consequently, a yellowspot indicts that the GFP may be in the same spot with the transferrinprotein, thus providing a reference to the location of GFP inside thecells. As shown in FIG. 10A-D, a significant number of green spots werevisible in cells treated with LFn-GFP for one hour, suggesting theLFn-GFP can indeed enter into cells.

FIG. 11 shows cellular uptake of GFP into CHO cells, using conditions asdescribed for FIG. 10. FIG. 11 A, D, and G show the results ofincubating CHO cells with GFP for 1 hour; FIG. 11 B-C, E-F, and H-I showthe results of incubating CHO cells with GFP for two hours. FIG. 11 A-Cshow fields of cells examined by confocal microscopy for green color(GFP); FIG. 11 D-F show the corresponding fields examined by confocalmicroscopy for red color (anti-transferrin), respectively. A third imageis presented for each field by super-exposing the green image with thered image of the same cells (FIG. 11 G-I). GFP-treated cells showed fewgreen spots (FIG. 11).

FIG. 12 shows co-localization of GFP and LAMP-2 immunofluorescence toindicate the lysosome, using the identical experimental proceduresdescribed above for FIG. 10. FIGS. 12 A-D show fields of cells examinedby confocal microscopy for red color (LAMP-2); FIGS. 12 E-H show thecorresponding fields examined by confocal microscopy for green color(LN-GFP). A third image is presented for each field by super-exposingthe green image with the red image of the same cells (FIGS. 12 I-L).

FIG. 13 shows co-localization of GFP and immunofluorescence to EEA-1 toindicate the endosome, using the identical experimental proceduresdescribed above for FIG. 10. FIGS. 13 A-D show fields of cells examinedby confocal microscopy for red color (EEA-1); FIGS. 13 E-H show thecorresponding fields examined by confocal microscopy for green color(LN-GFP). A third image is presented for each field by super-exposingthe green image with the red image of the same cells (FIGS. 13 I-L).

FIG. 14 shows co-localization of GFP and an anti-Golgi antibody, usingthe identical experimental procedures described above for FIG. 10. FIGS.14 A-B show fields of cells examined by confocal microscopy for redcolor (anti-Golgi antibody); FIGS. 14 C-D show the corresponding fieldsexamined by confocal microscopy for green color (LN-GFP). A third imageis presented for each field by super-exposing the green image with thered image of the same cells (FIGS. 14 E-F).

FIG. 15 shows co-localization of GFP and anti-20s immunofluorescence toindicate the proteosome. Conditions were similar to those used in FIG.10 except HeLa cells were uncubated with 40 μg/ml LNgfp for 2 hours.FIG. 15 A-D show fields of cells examined by confocal microscopy for redcolor (anti-20s antibody); FIG. 15 E-H show the corresponding fieldsexamined by confocal microbscopy for green color (LN-GFP), respectively.A third image is presented for each field by super-exposing the greenimage with the red image of the same cells (FIG. 15I-L). By comparingthe different images in FIGS. 12-15, the super-exposed images that havethe most yellow spots are those shown in FIG. 15 in which the greenspots representing the intracellular LFn-GFP overlap significantly withthe red spots representing cellular proteosome.

FIG. 16 shows that adding PA did not increase the number of green spotsinside cells in comparison with those in the absence of PA, underconditions similar to those used in FIG. 15. FIGS. 16 A-F showincubation of HeLa cells with LN-GFP in the absence of PA; FIGS. 16 G-Lshow incubation of HeLa cells in the presence of PA. FIGS. 16 A-B andG-H show fields of cells examined by confocal microscopy for red color(anti-20s antibody); FIGS. 16 C-D and I-J show the corresponding fieldsexamined by confocal microbscopy for green color (LN-GFP), respectively.A third image is presented for each field by super-exposing the greenimage with the red image of the same cells (FIGS. 16 E-F and K-L).

FIG. 17 Three groups of BALB/c mice, four in each group, were immunizedi.p. with different antigen formulations, respectively (15 μg LFn-p24plus 4 μg PA, 15 μg LFn-p24 plus 4 μg PA plus Alum, 15 LFn-p24 only plusAlum). At one-week post the immunization, splenic mononuclear cells fromimmunized BALB/c mice were used as the source of CTL. The CTL in thesplenic cultures were activated in vitro by culturing withgamma-irradiated and peptide-pulsed BALB/c splenocytes from un-immunizedanimals. After 6 days of culture in a 37° C. CO₂ incubator, mature CTL(effector cells) were tested for their ability to lyse either⁵¹Cr-labeled P20 peptide-pulsed P815 cells (positive targets) or⁵¹Cr-labeled medium-pulsed cells (negative targets). The percent lysisshown were subtracted from the background lysis of negative targets andpresented as the average of each group.

FIG. 18 Three groups of BALB/c mice, four in each group, were immunizedi.p. with 15 ug LFn-p24, 15 ug MLFn-p24, and 15 ug p24, respectively.The CTL in splenic tissues were tested one after the immunization.

DETAILED DESCRIPTION OF THE INVENTION

We have now discovered a method for delivering exogenous proteins to thecytosol, by binding a target antigen (such as a protein) to a transportfactor that contains a fragment of a bipartite protein exotoxin in theabsence of a protective antigen (PA).

Preferably, the target antigen is fused to the transport factor.Preferably, the transport factor is the protective antigen bindingdomain of lethal factor from B. anthracis, consisting of amino acids1-289 (SEQ ID NO:2) or a fragment thereof that does not contain the PAbinding domain such as the carboxy portion of SEQ ID NO:2. For example,a fragment of about the 80 carboxy-most amino acids of preferred groupof amino acid LFn 1 amino acid fragments for the transport factorcomprises at least 80 amino acids that show at least 80% homology to SEQID NO: 2 using Blast on default settings. Still more preferably, thefragment has at least 90% homology thereto, even more preferably, it hasat least 95% homology thereto. Preferably, that fragment does notcontain the PA binding domain. A preferred transport factor is the LFnfragment or a portion thereof. More preferably, the transport factorcomprises at least the 60 carboxy-most amino acids of LFn but not the PAbinding domain. One preferred fragment has about 105 amino acids or lessfrom the carboxy portion of SEQ ID NO:2.

The target antigen can include any molecule for which it would bedesirable to elicit a CMI response, including viral antigens and tumorantigens.

One embodiment of the invention provides a composition including thenovel fusion polypeptide for generating. Another embodiment of theinvention includes assays for measuring CMI responses by deliveringexogenous proteins to the cytosol. A preferred embodiment provides kitsfor measuring CMI responses.

Novel fusion polypeptides of the present invention comprise a transportfactor linked to a target antigen. The transport factor can include afragment of any bipartite protein exotoxin that delivers the protein tothe cytosol in the absence of protective antigen without including anyfragments that are toxic. This transport method is independent of the PAassociated pathway and thus PA is unnecessary. A preferred exotoxin isLethal Factor (LF) of B. anthracis (SEQ ID NO:1) (FIG. 2A).

The PA binding domain of LF, is part of LFn or a fragment thereof, LFnor a fragment thereof can be used to transverse a cell membrane, whereLFn consists amino acids 1-255 of LF (i.e. residues 34-289 of SEQ IDNO: 1) and corresponds to (SEQ ID NO:2) (FIG. 2B). Any fragment of LFncan be used as the transport factor. Preferably it does not contain thePA binding domain, which is present in the N-terminal half of LFn (aa1-149). One preferred fragment has 105 amino acids or less from thecarboxy portion of SEQ ID NO:2. Preferably, the 60 carboxy-most aminoacids, still more preferably the 80 carboxy-most amino acids. Apreferred transport factor is Fragment 3 (SEQ ID NO:3) (FIG. 2C).

One can also use other fragments as the transport factor. Preferably,the transport factor is 350 amino acids or less, still more preferably,300 amino acids or less, even more preferably, it is 250 amino acids orless.

Other fragments preferred as the transport factor have at least 55%homology to Fragment 3 of LFn (SEQ ID NO:3) (FIG. 2C). For example, afragment of Edema Factor of B. anthracis has approximately 57% homologyto Fragment 3_(g) using Blast on default settings. More preferably, ithas at least 65% homology thereof, still more preferably, it has atleast 75% homology thereto; even more preferably, it has at least 80%homology thereto; even more preferably, it has at least 90% homologythereto; even more preferably, it has at least 95% homology thereto.

The transport factor is linked to any antigen whose delivery to thecytosol is desired. Preferably, the linkage is a chemical linkage, forexample a peptide bond to form of a fusion protein. However, otherlinkers known in the art can be created. For example, a linker unit canbe part of the transporter that can then be chemically linked to thetarget antigen. Preferred antigens include viral, bacterial, parasitic,and tumor associated antigens. Preferred viral antigens include proteinsfrom any virus where a cell-mediated immune response is desired.Particularly preferred viruses include HIV-1, HIV-2, hepatitis viruses(including hepatitis B and C), Ebola virus, West Nile virus, and herpesvirus such as HSV-2. Preferred bacterial antigens include those from S.typhi and Mycobacteria (including M. tuberculosis). Preferred parasiticantigens include those from Plasmodium (including P. falciparum).

Preferred tumor antigens include those epitopes which are recognized ineliciting T cell responses, including but not limited to the following:prostate cancer antigens (such as PSA, PSMA, etc.), breast cancerantigens (such as HER2/neu, mini-MUC, MUC-1, HER2 receptor,mammoglobulin, labyrinthine, SCP-1, NY-ESO-1, SSX-2, N-terminal blockedsoluble cytokeratin, 43 kD human cancer antigens, PRAT, TUAN, Lbantigen, carcinoembryonic antigen, polyadenylate polymerase, p53, mdm-2,p21, CA15-3, oncoprotein 18/stathmin, and human glandular kallikrein),melanoma antigens, and the like.

Preferably, when one is trying to generate an immune response in asubject, an immune adjuvant is also used. Adjuvants are known in the artand include cytokines such as IL-2, Ig-IL-2, CM-CSF, CpG, RIBL Detox(Ribi Immunochemical), QS21 (Cambridge Biotech), incomplete Freund'sadjuvant and others. We have unexpectedly found that although Alum canactually inhibit CTL induction, in the present system, Alum is preferredfor stimulating specific CTL.

The methods of the present invention can also be used to identifyadditional cancer antigens, by making libraries of tumor antigens fusedto the transport factor in conjunction with the CMI assays, describedbelow.

The target antigen can be of any size that allows delivery to thecytosol. Preferably, the target antigen is less than 750 amino acids,still more preferably, less than 600 amino acids, even more preferably,less than 500 amino acids.

The novel fusion polypeptides of the present invention can include asingle target antigen or multiple target antigens as part of a singlefusion protein. A preferred fusion polypeptide includes fragments ofseveral HIV-1 proteins, such as gag and nef (FIGS. 8, 9).

One can also create epitopes using multiple strains of an infectiousvirus such as with HIV. (See FIG. 9).

The novel fusion polypeptides may be part of larger multimeric moleculeswhich may be produced recombinantly or may be synthesized chemically.Such multimers may also include the polypeptides fused or coupled tomoieties other than amino acids, including lipids and carbohydrates.

Preferably, the multimeric proteins consist of multiple T cell epitopesrepeated within the same molecule, either randomly, or with spacers(amino acid or otherwise) between them.

DNA sequences encoding these novel fusion proteins can readily be made.For example, the sequence encoding LFn is well known and can be modifiedby known techniques, such as deleting the undesired regions, such asvariable loops, and to insert any additional desired coding sequences,such linker segments. The sequences encoding various target antigens arealso known in the art. In addition, the codons for the various aminoacid residues are known and one can readily prepare alternative codingsequences by standard techniques.

DNA sequences can be used in a range of animals to express the novelfusion protein, which can then be used for a variety of uses, includingin a vaccine composition and in CMI assays, as described below.

DNA sequences encoding the novel fusion protein can be expressed in awide variety of host/vector combinations. Vectors include chemicalconjugates such as those described in WO 93/04701, which has targetingmoiety (e.g. a ligand to a cellular surface receptor), and a nucleicacid binding moiety (e.g. polylysine), viral vectors (e.g. a DNA or RNAviral vector), plasmids, phage, etc. The vectors can be chromosomal,non-chromosomal or synthetic.

Useful expression vectors for eukaryotic hosts, include, for example,vectors comprising expression control sequences from SV40, bovinepapilloma virus, adenovirus, adeno-associated virus, cytomegalovirus andretroviruses. Useful expression vectors for bacterial hosts includebacterial plasmids, such as those from E. coli, including pBluescript,pGEX-2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and their derivatives,wider host range plasmids, such as RP4, phage DNAs, e.g., the numerousderivatives of phage lambda, e.g. .lambda.GT10 and .lambda.GT11, andother phages. Useful expression vectors for yeast cells include the 2micron. plasmid and derivatives thereof. Useful vectors for insect cellsinclude pVL 941.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include Moloney murine leukemia virusesand HIV-based viruses. One preferred HIV-based viral vector comprises atleast two vectors wherein the gag and pol genes are from an HIV genomeand the env gene is from another virus. DNA viral vectors are preferred.These vectors include herpes virus vectors such as a herpes simplex Ivirus (HSV) vector (Geller, A. I. et al., J. Neurochem 64: 487, 1995;Lim, F. et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed.,Oxford Univ. Press, Oxford England, 199); Geller, A. I., Proc. Nat.Acad. Sci. USA 90: 7603, 1993; Geller, A. I., I Proc Natl. Acad. Sci USA87: 1149, 1990), adenovirus vectors (LeGal LaSalle et al., Science 259:988, 1993; Davidson, et al., Nat. Genet 3: 219, 1993; Yang, et al., J.Virol. 69: 2004, 1995), and adeno-associated virus vectors (Kaplitt, M.G., et al., Nat. Genet. 8:148, 1994). The DNA sequence is operablylinked to a promoter that permits expression in the host cell. Suchpromoters are well known in the art and can readily be selected.

A wide variety of unicellular host cells are useful in expressing theDNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fingi, yeast, insect cells such asSpodoptera frugiperda (SF9), animal cells such as CHO and mouse cells,African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40, and BMT10, and human cells, as well as plant cells in tissue culture. Themolecules of the present invention can be used to create a range ofproducer cells. This is particularly useful with certain proteins thatare currently difficult to express in cells in large amounts.

The molecules comprising the novel fusion polypeptides encoded by theDNA sequences of this invention may be isolated from the fermentation orcell culture and purified using any of a variety of conventional methodsincluding: liquid chromatography such as normal or reversed phase, usingHPLC, FPLC and the like; affinity chromatography (such as with inorganicligands or monoclonal antibodies); size exclusion chromatography;immobilized metal chelate chromatography; gel electrophoresis; and thelike. One of skill in the art may select the most appropriate isolationand purification techniques without departing from the scope of thisinvention.

Stabilized forms of these novel fusion proteins can readily be made, forexample, by conjugates such as a poly(alkylene oxide) conjugate. Theconjugate is preferably formed by covalently bonding the hydroxylterminals of the poly(alkylene oxide) and a free amino group in aportion of the fusion protein that will not affect its conformation.Other art recognized methods of conjugating these materials includeamide or ester linkages. Covalent linkage as well as non-covalentconjugation such as lipophilic or hydrophilic interactions can be used.

The conjugate can be comprised of non-antigenic polymeric substancessuch as dextran, polyvinyl pyrrolidones, polysaccharides, starches,polyvinyl alcohols, polyacryl amides or other similar substantiallynon-immunogenic polymers. Polyethylene glycol (PEG) is preferred. Otherpoly(alkylenes oxides) include monomethoxy-polyethylene glycolpolypropylene glycol, block copolymers of polyethylene glycol, andpolypropylene glycol and the like. The polymers can also be distallycapped with C1-4 alkyls instead of monomethoxy groups. The poly(alkyleneoxides) used must be soluble in liquid at room temperature. Thus, theypreferably have a molecular weight from about 200 to about 20,000daltons, more preferably about 2,000 to about 10,000 and still morepreferably about 5,000.

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant novel fusionproteins of the invention. See, for example, “Conjugate Vaccines”,Contributions to Microbiology and Immunology, J. M. Cruse and R. E.Lewis, Jr (eds.), Carger Press, New York, 1989, the entire contents ofwhich are incorporated herein by reference.

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the novel fusion protein and the other moietyretain their respective activities. This linkage can include manychemical mechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, disocyanates,glutaraldehydes, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents (see Killen and Lindstrom, J. Immunol. 133:1335-2549,1984; Jansen, F. K, et al., Imm. Rev. 62:185-216, 1982; and Vitetta etal., supra).

Preferred linkers are described in the literature. See, for example,Ramakrishnan, S., et al., Cancer Res. 44: 201-208 (1984), describing theuse of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See alsoUmemoto et al., U.S. Pat. No. 5,030,719, describing the use of ahalogenated acetyl hydrazide derivative coupled to an antibody by way ofan oligopeptide linker. Particularly preferred linkers include: (i) EDC(1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene(Pierce Chem. Co., Cat (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS(N-hydroxysulfosuccinimide: Pierce Chem. Co.,Cat #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

The novel fusion proteins of the present invention can be used for thestable expression of proteins. For example, certain proteins aredifficult to express in certain expression systems, including bacterialexpression systems. Fusion to a transport factor such as LFn canstabilize certain such proteins. We have found that the transport factoris preferably 250 amino acids or less, still more preferably 150 aminoacids or less, more preferably 105 amino acids or less, even morepreferably 80 amino acids or less.

The novel fusion proteins of the present invention can be used togenerate an immune response. For example, as a vaccine.

An exemplary pharmaceutical composition is a therapeutically effectiveamount of a novel fusion protein that can induce an immune reaction,thereby acting as a prophylactic immunogen, optionally included in apharmaceutically-acceptable and compatible carrier. The term“pharmaceutically-acceptable and compatible carrier” as used herein, anddescribed more fully below, includes (i) one or more compatible solid orliquid filler diluents or encapsulating substances that are suitable foradministration to a human or other animal, and/or (ii) a system, capableof delivering the molecule to a target cell. In the present invention,the term “carrier” thus denotes an organic or inorganic ingredient,natural or synthetic, with which the molecules of the invention arecombined to facilitate application. The term “therapeutically-effectiveamount” is that amount of the present pharmaceutical composition whichproduces a desired result or exerts a desired influence on theparticular condition being treated. For example, the amount necessary toraise an immune reaction to provide prophylactic protection. Typicallywhen the composition is being used as a prophylactic immunogen at leastone “boost” will be administered at a periodic interval after theinitial administration. Various concentrations may be used in preparingcompositions incorporating the same ingredient to provide for variationsin the age of the patient to be treated, the severity of the condition,the duration of the treatment and the mode of administration.

In a preferred embodiment, the novel fusion polypeptides of thisinvention which are also immunogenic polypeptides are incorporated intoa multicomponent vaccine which can comprise other immunogenicpolypeptides. A multicomponent vaccine may contain novel fusionprotein(s) of the present invention to elicit T cell responses, as wellas other antigens to elicit B cell responses.

In one preferred method of immunization one would prime with one novelfusion protein and then boost with a different novel fusion protein.

One can also use cocktails containing a variety of different novelfusion proteins to prime and boost with either a variety of differentnovel fusion proteins or with fusion proteins that contain multipleantigens (FIGS. 8, 9).

The novel fusion proteins can be used to generate a range of T cellsthat recognize and interact with a diverse range of antigens, forexample, from different HIV strains. The DNA sequence encoding the noelfusion proteins can also be used as a subunit vaccine.

In trying to generate an immune reaction such as with a vaccinecomposition, an adjuvant is preferably also used. Adjuvants include butare not limited to Alum, RIBI Detox (Ribi Immunochemical), QS21(Cambridge Biotech), and incomplete Freund's adjuvant. Alum is apreferred adjuvant. Another group of adjuvants include immunestimulators such as cytokines such as IL-12, IL-4 and costimulatorymolecules such as B7. A wide range of molecules having immunestimulating effects are known including accessory molecules such as ICAMand LFA. In a preferred embodiment GM-CSF is administered to the patientbefore the initial immune administration. GM-CSF may be administeredusing a viral vector or an isolated protein in a pharmaceuticalformulation. Combinations of adjuvants can be used such as CM-CSF, I CAMand LFA. While a strong immune response is typically generated toinfectious disease antigens, tumor associated antigens typicallygenerate a weaker immune response. Thus, immune stimulators such asdescribed above are preferably used with them. As aforesaid, Alum is apreferred adjuvant.

The immune stimulatory composition of the present invention may be usedadvantageously with other treatment regiments. For example, the systemmay be used in conjunction with traditional treatment options for cancerincluding surgery, radiation therapy, chemotherapy and hormone therapy.For example, a breast cancer vaccine comprising a novel fusion proteinof the present invention can be used in conjunction with tamoxifencitrate, which interferes with the activity of estrogen. The system mayalso be combined with immunotherapy, e.g. using Herceptin™(trastuzumab), an anti-HER2 humanized monoclonal antibody developed toblock the HER2 receptor; bone marrow transplantation; and peripheralblood stem cell therapy can also be used. Other preferred treatmentregiments to be used in conjunction with the present composition includeangiogenesis is inhibitors and cytotoxic agents.

The term “compatible”, as used herein, means that the components of thepharmaceutical compositions are capable of being commingled with a smallmolecule, nucleic acid and/or polypeptides of the present invention, andwith each other, in a manner such that does not substantially impair thedesired pharmaceutical efficacy.

Doses of the pharmaceutical compositions of the invention will varydepending on the subject and upon the particular route of administrationused. Dosages can range from 0.1 to 100,000 μg/kg per day, morepreferably 1 to 10,000 μg/kg. Preferred doses of the compositions arepreferably at least 2 μg/ml. By way of an example only, an overall doserange of from about, for example, 1 microgram to about 300 microgramsmight be used for human use. This dose can be delivered at periodicintervals based upon the composition. For example on at least twoseparate occasions, preferably spaced apart by about 4 weeks. Othercompounds might be administered daily. Pharmaceutical compositions ofthe present invention can also be administered to a subject according toa variety of other, well-characterized protocols. For example, certaincurrently accepted immunization regimens can include the following: (i)administration times are a first dose at elected date; a second dose at1 month after first dose; and a third dose at a subsequent date, e.g., 5months after second dose. See Product Information, Physician's DeskReference, Merck Sharp & Dohme (1990), at 1442-43. (e.g., Hepatitis BVaccine-type protocol); (ii) for example with other vaccines therecommended administration for children is first dose at elected date(at age 6 weeks old or older); a second dose at 4-8 weeks after firstdose; a third dose at 4-8 weeks after second dose; a fourth dose at 6-12months after third dose; a fifth dose at age 4-6 years old; andadditional boosters every 10 years after last dose. See ProductInformation, Physician's Desk Reference, Merck Sharp & Dohme (1990), at879 (e.g., Diphtheria, Tetanus and Pertussis-type vaccine protocols).Desired time intervals for delivery of multiple doses of a particularcomposition can be determined by one of ordinary skill in the artemploying no more than routine experimentation.

The novel fusion proteins of the invention may also be administered perse (neat) or in the form of a pharmaceutically acceptable salt. Whenused in medicine, the salts should be pharmaceutically acceptable, butnon-pharmaceutically acceptable salts may conveniently be used toprepare pharmaceutically acceptable salts thereof and are not excludedfrom the scope of this invention. Such pharmaceutically acceptable saltsinclude, but are not limited to, those prepared from the followingacids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic,acetic, salicylic, p-toluene-sulfonic, tartaric, citric,methanesulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, andbenzenesulphonic. Also, pharmaceutically acceptable salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group. Thus, thepresent invention also provides pharmaceutical compositions, for medicaluse, which comprise nucleic acid and/or polypeptides of the inventiontogether with one or more pharmaceutically acceptable carriers thereofand optionally any other therapeutic ingredients.

The compositions include those suitable for oral, rectal, intravaginal,topical, nasal, ophthalmic or parenteral administration, all of whichmay be used as routes of administration using the materials of thepresent invention. Other suitable routes of administration includeintrathecal administration directly into spinal fluid (CSF), directinjection onto an arterial surface and intraparenchymal injectiondirectly into targeted areas of an organ. Compositions suitable forparenteral administration are preferred. The term “parenteral” includessubcutaneous injections, intravenous, intramuscular, intrasternalinjection or infusion techniques. Intramuscular administration ispreferred.

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.Methods typically include the step of bringing the active ingredients ofthe invention into association with a carrier which constitutes one ormore accessory ingredients.

Compositions of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets, tablets orlozenges, each containing a predetermined amount of the nucleic acidand/or polypeptide of the invention in liposomes or as a suspension inan aqueous liquor or non-aqueous liquid such as a syrup, an elixir, oran emulsion.

Preferred compositions suitable for parenteral administrationconveniently comprise a sterile aqueous preparation of the molecule ofthe invention which is preferably isotonic with the blood of therecipient. This aqueous preparation may be formulated according to knownmethods using those suitable dispersing or wetting agents and suspendingagents. The sterile injectable preparation may also be a sterileinjectable solution or suspension in a non-toxic parenterally-acceptablediluent or solvent, for example as a solution in 1,3-butane diol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

To further improve the likelihood of producing a cell mediated responseas provided by the invention, the amino acid sequence of polypeptidesencoded by a nucleotide sequence of the present invention may beanalyzed in order to identify desired portions of amino acid sequencewhich may be associated with receptor binding. For example, polypeptidesequences may be subjected to computer analysis to identify such sites.

Complexes that form with molecules of the present invention can bedetected by appropriate assays, such as the CMI assays discussed belowand by other conventional types of immunoassays.

The present invention provides assays to measure cell-mediated immuneresponses in vitro. Current available laboratory assays to detect CMIresponses have serious shortcomings, especially when applied to largevaccine efficacy trials at various clinical settings. This is becausethe available equipment and technical support are minimal. As discussedabove, the belief that CTL play a crucial role in controlling HIV-1infection, many HIV-1 vaccine candidates are designed to stimulate Tcell responses as well as neutralizing antibodies (1-6).

The method of the present invention for measuring T cell responses willhave a significant effect on the development of all T cell dependentvaccines or immune therapies. This strategy is applicable to other fieldof research where CMI responses play an important role in prevention andcontrolling of the diseases.

In a preferred embodiment, the present invention provides use of thenovel fusion polypeptides in the Elispot assay. The main advantage ofthe Elispot assay is that a large number of CTL responses can beevaluated effectively and efficiently in a large number of people (seefor example, Lalvani et al., J. Exp. Med. 186:859-65 (1997)). TheElispot assay can use any standard cells, including cryopreserved PBMCsas well as CD8+ CTL cells. For example, CD8+ CTL clones from HIV-1positive individuals.

Another preferred embodiment provides use of the novel fusionpolypeptide in flow-based intracellular cytokine assays. Flowcytometry-based assays detect the intracellular accumulation ofcytokines after in vitro stimulation of antigen specific T cells. CTLsare stimulated with antigens and incubated with brefeldin A, whichinhibits protein transport and allows intracellular accumulation ofnewly synthesized proteins such as cytokines. Surface markers (CD8, CD3)and intracellular staining of IFN-γ and CD69 allows detection andquantitation of specific cell populations against antigenic epitopes inthe context of uncharacterized MHC backgrounds.

The CMI assays using the novel fusion polypeptides of the presentinvention can be used to evaluate cellular immune responses against abroad array of viral, bacterial, and tumor antigens. The assays can beused in either evaluating vaccine efficacy or in arresting the statuesof infection by infectious pathogens.

The CMI assays using the novel fusion polypeptides of the presentinvention can also be used as early diagnostic kits to detect cancerantigens.

The present invention also provides a kit for detecting CMI responses.One preferred kit contains novel fusion proteins in these tubes, wells,etc. The kit preferably contains viral or tumor containing reagents todetect CMI responses in vitro. Preferably, the kit contains an apparatusfor removing a biological specimen such as blood. In a preferredembodiment, the kit will contain instructions for detecting CMIresponses. Preferably, the kit contains the novel fusion protein in alyophilized form.

A preferred kit includes the novel fusion protein lyophilized and coatedonto the inside of a tube used for the collection of blood. This allowsblood to be drawn from an individual directly into the tube, incubatedfor several hours (for example, 4 hours), and then fixed. Such fixedsamples are stable for use in CMI assays for extended periods of time(for example, two weeks). The kit is particularly useful for correctingsamples in the field, for example, during vaccine efficacy trials, whichtypically occur in remote locations. Similarly, other preferredembodiments include the lyophilized protein coated onto any surface,such as a dish, a well, or a tube, which is amenable to adding a smallvolume of a biological specimen. Preferred biological specimens includeblood, urine, sputum, stool, supernatant from cerebrospinal fluid, andcell samples.

In a specific example, LFn fusion proteins such as those described abovewhich can contain only the carboxy portion of LFn, can in the absence ofPA sensitize CTL target cells in a MHC-I restricted manner. In thisnovel method of presenting exogenous protein antigens to the MHC-Ipathway, the cytosolic delivery of the antigens relies on the functionaltransportation associated with intracellular antigen processing, withoutresorting to live viral or bacterial vectors. These LFn fragments are anexample of a new class of proteins that enters the MHC-I pathway fromthe cytosol. The fact that for example, LFn can deliver HIV-1 antigensinto cytosol in the absence of PA is in contrary to the currenthypothesis of how anthrax lethal toxin works (32). In the normal case,LF (anthrax lethal factor) is dependent on PA to exert its toxic effectand possibly the toxic domain of LF possess unknown functions thatprevent cell entry.

Additionally, fusion proteins such as LFn-HIV can be used as a tool torefine and simplify the methods for detecting T cell responses invaccine trials. This method is also useful for studies of otherintracellular viral or bacterial diseases, such as viral hepatitis, TB,and malaria. The present invention can circumvent the need for usinglive recombinant vaccinia viruses in the current CMI assays. It alsocarries several advantages over using overlapping synthetic peptides,which are not only expensive, but that their theoretical coverage ofdifferent populations with HLA-I diversity remains unproven. The novelantigen delivery provides simplified CMI assays that are widelyapplicable to field use. Furthermore, these LFn fusion proteins areeasily produced in E. coli, purified in relatively simple procedures,and are stable.

All of the novel fusion polypeptides provided by this invention, and theDNA sequences encoding them, are substantially free of a B. anthracisprotective antigen or fragments thereof, and thus may be safely used ina variety of applications without the risk of unintentional damage tothe host. Accordingly, the novel fusion polypeptides of this inventionare particularly advantageous in compositions and methods for generatinga broad range of cell-mediated responses to target antigens.

The following Examples serve to illustrate the present invention, andare not intended to limit the invention in any manner.

Example 1 Materials and Methods

LFn-HIV Fusion Proteins

The DNA fragment encoding env gp120, gag p24 from HIV-HXB were amplifiedby PCR, cloned into the LFn expression plasmid pET15bLFn, and sequencedto verify the inframe fusion between the LFn and the HIV codingsequence. The Nef coding sequence was amplified from HIV-ELI. Theprotein expression vector for LFn and its fusion derivatives is thepET15b plasmid (Novagen; Madison, Wis.). The main features of thisvector system include an inducible T7 promoter, an internal His-Tag forprotein purification, and multiple cloning sites. The recombinant LFn isexpressed in E. coli as an intracellular soluble protein with 6 tandemhistidine residues at its N-terminal end (14). Bacteria were grown in aten-liter Bioflow 2000 bench top bioreactor (New Brunswick Scientific,NJ). Purification of the His-tagged proteins was conducted using acommercial kit according to the manufacturer's protocol (Novagen).Fragments of LFn can be made by modifying the LFn coding region by knowntechniques.

Reagents

Synthetic peptides were synthesized on an Applied Biosystems PeptideSynthesizer (Model 430A). Recombinant vaccinia viruses used were vAbT141 (Gag), vAbT 299 (Env), and Nef (15) with NYCH as control vacciniavector. Brefeldin A, cytochalasin B and chloroquine (Sigma, St Louis,Mo.) were added to cell culture for 2 hours and washed twice beforeadding back to the cells. Antibodies for flow analysis were obtainedfrom Becton-Dickinson (San Jose, Calif.)

Flow-Based Intracellular Cytokine Staining

Cryopreserved PBMCs were incubated overnight with LFN-HIV (30 ug/ml),recombinant vaccinia vectors (MOI 3-5) or peptides (10 ug/ml) at 37° C.in 5% CO₂. Autologous B-LCLs were incubated overnight with optimalpeptides (10 ug/ml), LFn-HIV (10 ug/ml) or recombinant vaccinia viruses(MOI 3-5), washed twice and added to effector cells at E:T ratio of 10:1in the presence of co-stimulatory anti-CD28 and anti-CD49d (1 ug/ml,Becton-Dickinson, San Jose, Calif. Brefeldin A (10 ul of 1 mg/ml) wassubsequently added and the cell culture was incubated at 37° C. for 6hours and stained with saturating solution of isotype controls (IgG1 forAPC, PE, PerCP and IgG2b for FITC) or allophycocyanin (APC)-labeled CD3monoclonal antibody (Mab), phycoerylrin (PE)-labeled anti-CD8 Mab(Becton Dickinson). After incubating in dark at 4° C. for 20 minutes,the cells were washed twice with FACS wash. 100 ul of reagent A (Fix andPerm kit, Caltag Laboratories, Austria) was added and after incubatingin dark at room temperature (RT) for 20 minutes, the cells were washedtwice with FACS wash. 100 ul of reagent B (Fix and Perm kit) was thenadded and cells were incubated at RT for 5 minutes. After addingFITC-IFN gamma and peridinin chlorophyll protein (PerCP)-conjugated CD69Mab (CD69 perCP) antibody and incubating in dark at 4° C., the cellswere washed twice with FACS wash and analyzed on Becton DickinsonFACScalibur flow cytometer using the Cell Quest software.

Samples were analyzed with four colors staining using the FACSCaliburFlow Cytometer (Becton Dickinson) and Cellquest software (BectonDickinson). Negative and positive controls used were unstimulated cellsas well as cells stimulated, respectively, with mitogenphematohemaglutinin PHA (0.25 ug/ml, Murex Biotech).

Cell Lines and Culture Conditions.

Autologous Epstein-Barr virus (EBV)-transformed B lymphoblastoid as wellas T1 and T2 (HLA-B60) cell lines were used as antigen presenting cells(APCs). Target cells were pulsed with the appropriate index peptides orLFn-HIV constructs or infected overnight with recombinant vacciniaviruses. AC13 (HLA B14 restricted p24 [DRFYKTLRA]) (SEQ ID NO: 4) and KM(HLV B60-restricted Nef [KEKGGLEGL]) (SEQ ID NO: 5) are Gag andNef-specific clones (respectively) and AC2 (HLA B44 restricted gp120[AENLWVTVY]) (SEQ ID NO: 6). All were obtained from HIV infectedindividuals whose CTL responses are well characterized (Rosenberg,personal communication) and the Env-specific clone (SP 511) wasgenerated from a seropositive Ugandan (16). PMBCs used in the Elispotstudy were HIV-I infected individuals from Senegal and Uganda (15, 16).To investigate the effect of brefeldin A, cytochalasin B or chloroquineon MHC class I restricted presentation of LFn-HIV, APCs were labeledwith the protein constructs, cultured in RPMI 1640 m 10% FCS andcocultured with respective reagents for one hour, then washed and testedin the standard chromium release assay.

Elispot

Elispot assay was performed on cryopreserved PBMCs. 96-wellnitrocellulose plates (Millititer, Millipore Corp., Bedford, Mass.) werepre-coated overnight in 4° C. with 0.5 mg/ml of monoclonal antibody1-D1K (Mabtech, Stockholm, Sweden). The plates were then washed 6× withphosphate-buffered saline (PBS) and PBMCs were added at 50,000cells/well and 25,000 cells/well in duplicate wells, respectively. Theplates were incubated overnight at 37° C. in 5% CO2 and biotinylatedmonoclonal antibody anti-IFN-Mab (Mabtech) was then added at 0.5 mg/mlfor 100 minutes followed by streptavidin-ALP (Mabtech) for 1 hour atroom temperature. The plates were washed three times with PBS and5-bromo-4-chloro-3-indolyl phosphate and nitro blue (Sigma) added todevelop the reaction. Tap water was added to stop the reaction after 15minutes. Individual cytokine-producing cells were detected as darkspots, which were visualized and quantitated as SFC/well (spot formingcolony/well). CTL frequency (CTLp) was calculated from the number ofspots subtracted from the control wells and averaged from the fourwells. The final CTLp was reported as the average frequency per 10⁶cells. Responses were considered positive if the SFCs were at leasttwice that of control. Background SFC was on average less than 15/well.

Chromium Release Assay:

CD8+ CTL clones were stimulated with anti CD3 monoclonal antibody (12F6)in the presence of recombinant IL-2 and tested for activity within sevendays. Target cells included autologous B-LCL or HLA-matched APCsinfected with recombinant vaccinia virus (multiplicity of infection 3 to5) expressing HIV-1 gene products or with LFn proteins (5 to 30 ug/ml)overnight and labeled with radioactive chromium (⁵¹Cr). Effector:targetcell ratio (E:T) was 10:1 in a final volume of 200 ul with all assaysperformed in duplicate wells. Supernatant fluid was harvested after 4hours and the percent specific lysis was determined from the formula:100×[(experimental release−spontaneous release)/(maximumrelease−spontaneous release)]. HIV-specific CTL activity was defined as10% above background/control. Spontaneous release was <30% of maximalrelease for all assays.

LFn-HIV Mediates HIV-1 Antigen Entry and Processing without PA:

To assess the ability of LFn-HIV to associate with MHC-1 molecule oncell surface, lymphoblastoid B cell lines (B-LCL) were sensitized withLFn-HIV in the presence or absence of PA and tested in a standardchromium release assay using several well characterized HIV-specific CTLclones (FIG. 3). We found that CTL recognized target cells sensitizedwith LFn-HIV comparably in the presence or absence of PA. Lysis levelwas dose-dependent and achieved to levels comparable to positivecontrols where B-LCLs were infected with recombinant vaccinia virusesexpressing the same HIV antigens. HIV-specific CTL activity was evidentonly when targets had a longer incubation period with LFn-HIV (8 hoursvs 1 hour) (data not shown), suggesting a delay in the appearance ofsurface antigen and the requirement for intracellular processing.

LFn-HIV is Presented in the Classical MHC-1 Pathway

We next verified that LFn-HIV was presented by the classical MHC-Ipathway, by demonstrating that the recognition of these constructs byCTLs was HLA-I restricted (FIG. 4). Two Nef- (KM) and Gag-specific(AC13) clones that are HLA B60- and B14-restricted, respectively, weretested with HLA matched and mismatched targets presenting LFn-Nef orLFn-p24. Only HLA-matched targets demonstrated significant lysis,confirming the MHC-I presentation of LFn-HIV.

We further examined the potential mechanism of uptake and processing ofLFn-HIV. To determine whether the internalization of LFn-HIV at thesurface was actively processed, we pre-incubated B-LCL with 100 uM ofcytochalasin B (17, 18), a phagocytosis inhibitor before adding LFn-HIVantigens. CTL recognition was abrogated in the presence of cytochalasinB, suggesting an endocytosis-mediated internalization process.

If exogenous LFn fusion protein was introduced and subsequentlyprocessed in the cytosol, TAP proteins (Transporter associated withantigen processing) would be required for the transport of peptides intothe ER lumen for binding to MHC-I (19-21). To evaluate the requirementfor antigenic processing after endocytosis, we tested the ability ofLFn-HIV fusion proteins to sensitize the B60-expressing cell lines T1and T2 to lysis by CTL. T2 cell (22-24) is a derivative of T1 cellslacking the function of TAP, which transports peptides from theproteosome to the ER lumen for binding to MHC-I (19, 20). Recognitionwas abrogated in T2 cells (FIG. 4B), indicating the need for epitopetransport after processing. Recognition of this TAP deficient target bythe same clone was maintained if the cells were pulsed with the optimalepitope peptide directly to the surface “empty” MHC-I molecule,bypassing the requirement for intracellular processing.

To further verify the mechanism of exogenous protein entry into the MHCclass I pathway, target cells were treated with brefeldin A, whichinhibits exocytosis of proteins from the ER and Golgi complexes andprevents newly assembled peptide-MHC complexes from reaching the cellsurface (25). The addition of brefeldin A before presenting the targetcells with LFn-HIV effectively blocks recognition by CTLs (FIG. 5). Thisfinding, together with the earlier demonstration for HLA restriction andTAP requirement, confirms that LFn-HIV was processed and presented byB-LCL in a MHC class I pathway.

Processing of LFn-HIV is Chloroquine Insensitive

The presentation of many exogenous antigens on MHC-I molecule involvesproteolysis in the endocytic compartment (26-28) and the peptidessubsequently gain access to the cytosol where it enters the MHC class Ipathway. To investigate whether LFn mediated antigen presentationrequires proteolysis in acidic vesicles, we treated the B-LCL withchloroquine during exposure to LFn-HIV (FIG. 5). It is known thatchloroquine raises the pH in the endosomal and lysososmal compartmentsthus inhibiting protein hydrolysis by cathepsins, which require anacidic environment for activity (29, 30). Our finding suggested thatphagosomes with leaky properties are not requisite for the entry ofLFn-HIV into the cytosol.

The addition of either cytochalasin B, brefeldin A or chloroquine didnot affect recognition and lysis of target cells if targets were pulsedwith the optimal 8-mer epitopes at the surface, demonstrating that thesereagents did not affect CTL function nor target viability (FIG. 5).

Comparing LFn-HIV with Recombinant Vaccinia Virus in Different CMIAssays

Having demonstrated the ability of LFn-HIV to present CTL epitopes withMHC-I molecules at the cell surface, we proceeded to test theapplicability of the LFn fusion proteins in various CMI assays,including the enzyme linked spot (Elispot) assay and flow-basedintracellular interferon assay. These assays provide simpler and morerapid estimate of T cell responses and theoretically better suited forlarge clinical testing, especially in context of vaccine studies. Themain advantage of the Elispot assay is that the HIV-specific CTLresponse can be evaluated effectively and efficiently in large number ofpeople (31). Currently various antigen stimuli are used in this assay,including recombinant vaccinia viruses expressing HIV antigens oroverlapping synthetic peptides. Cryopreserved PBMCs as well as CD8+ CTLclones from several HIV-1 positive individuals were tested in theElispot assay, using LFn-p24, LFn-Nef or LFn-gp120 in parallel withrecombinant vaccinia viruses expressing the same antigens. Comparablespot forming colonies were observed with LFn-HIV and in some instances,less background spots was evident when compared to that with recombinantvaccinia virus (FIG. 6).

Flow cytometry-based assay detects the intracellular accumulation ofcytokines after in vitro stimulation of antigen-specific T cells. CTLsare stimulated with antigens and incubated with brefeldin A, whichinhibits protein transport and allow intracellular accumulation of newlysynthesized cytokines such as IFN-γ. Surface markers (CD8, CD3) andintracellular staining of IFN-γ and CD69 allows detection of andquantification of specific T cell population against antigenic epitopesin the context of uncharacterized HLA-I backgrounds. A Nef-specificclone (KM) was stimulated with LFn-Nef and subsequently evaluated forintracellular production of the interferon by flow analysis (FIG. 7A).Nef-specific response was detected with LFn-Nef as well as recombinantvaccinia virus expressing Nef in a cell population that is CD8+. Freshlyisolated, unstimulated PBMCs from an individual (AC2) with known gp120epitope was sensitized with LFn-Env as well as with recombinant vacciniavirus expressing Env (rVV-Env). The percentage of intracellular IFN-γ inthe CD8+ cell population was higher in cells exposed to LFn-Env comparedto those incubated with rVV-Env (FIG. 7B).

Example 2

LFn fusion proteins in the absence of PA are also capable of sensitizingCTL target cells in a MHC-I restricted manner. This cytosolic deliveryof LFn fusion proteins relies on functional transportation associatedwith intracellular antigen processing inside an antigen-presenting cell(Cao H, Agrawal D, Kushner N, Touzjian N, Essex M, and Lu Y. J. Infect.Dis. 185:244-251 (2002)). We have now demonstrated that GreenFlorescence Protein (GFP) fused with LFn can enter into cells in theabsence of PA. We further show that this PA-independent LFn delivery ofexogenous GFP appears to be associated with cellular proteosome, whichis consistent with the previous observation.

Construction and Purification of LFn-GFP and GFP

LFn-GFP fusion protein was constructed by insertion of the GFP openreading frame from pEGFP-C1 (Clontech) into the LFn expression vectordescribed in previous studies (Lu, Y., R. et al., Proc. Natl. Acad. Sci.USA 97:8027-32 (2000)). The fusion protein is soluble in bacterial cellextract and can be purified in one step affinity chromatography. Thepurified fusion protein has a molecular weight of approximately 55 kDand its solution has a bright green color (data not shown). In order tohave an appropriate control for the experiments, GFP alone wasconstructed into the same bacterial expression vector, pET15b, so thatthe only difference in the expression, purification, and use of GFP andLFn-GFP is the lack of the LFn sequences in GFP.

LFn-GFP Enters CHO Cells Whereas GFP Cannot

CHO cells were cultured in collagen treated chamber slides to reach 80%confluence and were then incubated with purified LFn-GFP for one or twohours, followed by washing extensively with PBS and PBS plus proteasesto remove membrane bound proteins. The cells were then stained with theanti-transferrin antibodies and fixed with paraformaldehyde. The slideswere examined by confocal microscopy for green color (GFP) and red color(anti-transferrin), respectively. A third image is presented for eachfield by super-expose the green image with the red image of the samecells. Consequently, a yellow spot indicts that the GFP may be in thesame spot with the transferrin protein, thus providing a reference tothe location of GFP inside the cells. As shown in FIG. 10, a significantnumber of green spots were visible in cells treated with LFn-GFP for onehour, suggesting the LFn-GFP can indeed enter into cells. Under theexactly same experimental conditions, GFP-treated cells showed few greenspots (FIG. 11).

Location of LFn-GFP Inside Treated-Cells

Using the identical experimental procedures described above for FIGS.10-11, we substituted the anti-transferrin antibodies with ananti-lysosome antibody (FIG. 12), an anti-endosome antibody (FIG. 13),an anti-Golgi antibody (FIG. 14), and an anti-proteosome antibody (FIG.15), respectively. By comparing these different images, thesuper-exposed images that have the most yellow spots are those shown inFIG. 15 in which the green spots representing the intracellular LFn-GFPoverlap significantly with the red spots representing cellularproteosome.

The Presence of PA does not Enhance the Cellular Intake of LFn-GFP

We further examined if the presence of PA would enhance the intake ofLFn-GFP under the experimental conditions used for FIGS. 10-15. As shownin FIG. 16, adding PA did not increase the number of green spots insidecells in comparison with those in the absence of PA. Moreover, it seemsthat the presence of PA might have reduced the amount of yellow spots.Apparently, it is likely that under these conditions these yellow spotsstill represent the PA-independent entry of LFn-GFP.

Example 3 Deletion of the N-Terminal Half of LFn, and the AdjuvantEffect of Alum on LFn Immunization

Additional animal immunization data demonstrates that the N-terminalhalf of LFn, which contains the PA binding domain, can be deleted withno negative effect on the antigen delivery. Quite unexpectedly, we foundthat the addition of Alum could significantly enhance the CTL inductionby the LFn fusion proteins.

Despite many unsuccessful attempts in the past two years, we have beenunable to show that LFn-p24 in the presence of PA could stimulate CTL inimmunized BALB/c mice. Previous studies have shown that LFn fusionproteins such as LFn-V3, LFn-LLO, and LFn-OVA are capable of stimulatingspecific CTL in mice (Lu, Y., et al. Proc. Natl. Acad. Sci. USA97:8027-32 (2000); Ballard, J. D. et al., Proc. Natl. Acad. Sci. USA 93,12531-12534 (1996); Ballard, J. D., et al., Infect. Immun. 66, 615-619(1998)). These fusion proteins carry only 12 to 33 amino acids, whereasLFn-p24 carries about 230 amino acids. Thus the size of the insertedantigens may make the difference. We decided to test if adding immuneadjuvants could improve the immunogenicity of LFn-p24. Among thereagents we tested, which include recombinant IL2, Ig-IL2, CpG, Alum,and others, Alum unexpectedly showed the best adjuvant activity. It iswidely believed that certain experimental adjuvants, such as QS21 andPCPP can enhance cell-mediated immune responses, whereas Alum actuallyinhibits the CTL induction (Schiembeck, R., et al., J. Immunol.152:1110-1119 (1994); Barouch, D. H. et al, Science 290:486 (2000);Davis, H. L. et al., J. Immunol. 160:870-876 (1998); Payne, L. G. etal., Dev. Biol. Stand. 92:79-87 (1998)).

In FIG. 17, three groups of BALB/c mice, four in each group, wereimmunized i.p. with different antigen formulations, respectively (15 ugLFn-p24 plus 4 ug PA, 15 ug LFn-p24 plus 4 ug PA plus Alum, 15 LFn-p24only plus Alum). At one-week post the immunization, splenic mononuclearcells from immunized BALB/c mice were used as the source of CTL. The CTLin the splenic cultures were activated in vitro by culturing withgamma-irradiated and peptide-pulsed BALB/c splenocytes from un-immunizedanimals. After 6 days of culture in a 37° C. CO₂ incubator, mature CTL(effector cells) were tested for their ability to lyse either⁵¹Cr-labeled P20 peptide-pulsed P815 cells (positive targets) or⁵¹Cr-labeled medium-pulsed cells (negative targets). The percent lysisshown were subtracted from the background lysis of negative targets andpresented as the average of each group.

As shown in FIG. 17, with the addition of Alum, we were able to showthat LFn-p24 can stimulate specific CTL in immunized mice in thepresence or absence of PA. These results further demonstrate that thepresence of PA is not required for the antigen delivery in vivo.

LFn Fusion Proteins where the N-Terminal Half of LFn has been Deletedare Still Active In an effort to confirm that LFn-p24 was able to elicitCTL in mice in the absence of PA, we constructed a mutated LFn fusionprotein (MLFn-p24) in which 1-149 amino acids at the N-terminal of LFnwas deleted to abolish its ability to bind PA. The mutated LFn (MLFn),with the deletion of 1-149 N-terminal amino acids from LFn (LFn is 1-255amino acids), results in a C-terminal amino acid fragment of LFn termedherein as fragment 3 (SEQ ID NO:3) (FIG. 2C). We confirmed this by usingan experimental method specially designed to test PA-dependent membranetranslocation. In brief, trypsin-nicked PA was incubated with CHO-K1cells for 2 hours at 4° C. The cells were then washed with cold PBS andincubated with ³⁵S-labeled LFn-NG or LFn-ENV for 2 hours at 4° C. Thecells were washed extensively and exposed to MES/gluconate buffer (pH4.8) at 37° C. for 2 minutes. Pronase E or buffer was then added todigest surface bound LFn fusion proteins that had not been internalized.The cells were then washed again, lysed, and counted. The percentage ofthe protein translocated by PA was calculated according the followingformula. The ratio=100×(counts in the presence of PA and the Pronasetreated cells−counts in the absence of PA and the Pronase treatedcells)/(counts in the presence of PA and in mock treated cells−counts inthe absence of PA and the mock treated cells). In this particular assay.PA translocated as high as 72% of the membrane bound LFn into the cells,whereas the amount of the MLFn translocated by PA was undetectable.

We then compared the CTL induction by LFn-p24 with MLFn-p24 in immunizedmice. In FIG. 18, three groups of BALB/c mice, four in each group, wereimmunized i.p. with 15 μg LFn-p24, 15 μg MLFn-p24, and 15 μg p24,respectively. The CTL in splenic tissues were tested one after theimmunization.

As shown in FIG. 18, both LFn-p24 and MLFn-p24 stimulated significantCTL activity after only one immunization. In fact, we repeatedlyobserved an improved CTL induction by MLFn-p24 compared to that inducedby LFn-p24. This experiment also demonstrates that the C-terminal halfof LFn (150-253) is indeed responsible for the intracellular antigendelivery, as the deletion of the MLFn sequence from the antigen (p24)abolishes the efficient CTL induction.

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All references described herein are incorporated herein by reference.

1. A method of delivering a target antigen to the cytosol of a cell,comprising binding the target antigen to a transport factor, wherein thetransport factor comprises a fragment of at least residues 185-288 ofSEQ ID NO: 2 and wherein a protective antigen (PA) from the bipartiteexotoxin from B. anthracis is not used, and wherein the protectiveantigen binds to a mammalian cell's surface by cellular receptors; andcontacting the cell with the target antigen bound to the transportfactor in the presence of an immune adjuvant selected from the groupconsisting of Alum, Incomplete Freund's Adjuvant, QS21, CpG, and RIBIDetox, thereby delivering the target antigen to the cytosol of the cell.2. The method of claim 1, wherein the transport factor does not containamino acids 1-184 of SEQ ID NO:2.
 3. The method of claim 1, wherein thetransport factor is SEQ ID NO:
 3. 4. The method of claim 1, wherein thetransport factor is 350 amino acids or less.
 5. The method of claim 1,wherein the transport factor is 300 amino acids or less.
 6. Apharmaceutical composition comprising an immunogenic amount of theisolated polypeptide of claim
 5. 7. The pharmaceutical composition ofclaim 6, wherein the composition further contains a co-stimulatorymolecule or a non-antigenic polymeric substance conjugated to saidtarget antigen or said transport factor.
 8. A kit for measuring cellmediated immune responses in vitro, comprising the novel polypeptide ofclaim
 5. 9. The method of claim 5, wherein the transport factor is LFnpolypeptide which comprises residues 34-288 of SEQ ID NO:
 2. 10. Themethod of claim 1, wherein the transport factor is 250 amino acids orless.
 11. The method of claim 1, wherein the target antigen is selectedfrom the group consisting of viral antigens, bacterial antigens, andtumor antigens.
 12. The method of claim 11, wherein the viral antigen isa Human Immuno-deficiency virus (HIV) antigen.
 13. The method of claim1, wherein the transport factor is bound to the target antigen byexpression of a fusion polypeptide wherein a single nucleic acid codingsequence encodes both the transport factor and the target antigen. 14.The method of claim 1, wherein the transport factor is bound to thetarget antigen by a chemical linkage.
 15. The method of claim 1, whereinthe transport factor comprises residues 34-288 of SEQ ID NO:
 2. 16. Apharmaceutical composition and an adjuvant, comprising an immunogeniceliciting amount of a transport factor bound to a target antigen,wherein the transport factor comprises at least residues 185-288 of SEQID NO: 2, and wherein a protective antigen (PA) from the bipartateexotoxin from B. anthracis is not used and wherein the adjuvant is Alum.17. A method of delivering a target antigen to the cytosol of a cellcomprising contacting the cell with a composition comprising a targetantigen bound to a transport factor and at least an immune adjuvantselected from the group consisting of Alum, incomplete Freund'sAdjuvant, Qs21, CpG, and RIBI Detox a co-stimulatory molecule or anon-antigenic polymeric substance conjugated to said target antigen orsaid transport factor, wherein the transport factor comprises at leastamino acids 185-288 of SEQ ID NO: 2 and is not toxic to the cells, andwherein a protective antigen (PA) that is part of a bipartite proteinfrom B. anthracis is not present.
 18. The method of claim 17, whereinthe transport factor is LFn polypeptide and corresponds to residues34-288 of SEQ ID NO:
 2. 19. The method of claim 17, wherein thecomposition is administered to a subject to stimulate the immune system.20. A method of delivering a target antigen to the cytosol of a cell,comprising binding the target antigen to a B. anthracis LFn or the LFnfragment, wherein LFn comprises residues 34-288 of SEQ ID NO: 2 andwherein a LFn fragment is at least Fragment 3 (SEQ ID NO: 3), andcontacting the cell in the presence of Alum, but in the absence of a B.anthracis protective antigen (PA) wherein the protective antigen is partof the bipartite protein, and wherein the target antigen bound to theLFn or LFn fragment is delivered to the cytosol of the cell.
 21. Themethod of claim 20, wherein the transport factor corresponds to SEQ IDNO:3.